Novel porous biomaterials

ABSTRACT

The invention provides porous biomaterials and methods for forming porous biomaterials. The porous biomaterials of the invention comprise a biocompatible polymer scaffold defining an array of pores, wherein substantially all the pores have a similar diameter, wherein the mean diameter of the pores is between about 20 and about 90 micrometers, wherein substantially all the pores are each connected to at least 4 other pores, and wherein the diameter of substantially all the connections between the pores is between about 15% and about 40% of the mean diameter of the pores. The invention also provides implantable devices comprising a layer of a biomaterial, and methods for promoting angiogenesis in and around an implantable biomaterial.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.60/507,734, filed Oct. 1, 2003.

GOVERNMENT RIGHTS

This invention was made with government support under EEC-9529161awarded by the National Science Foundation and under R24HL 64387 awardedby the National Institutes of Health. The government has certain rightsin the invention.

FIELD OF THE INVENTION

The present invention relates, to porous biomaterials that support thein-growth of blood vessels, and implantable devices comprising them. Theinvention also relates to methods for forming and using the porousbiomaterials of the invention.

BACKGROUND OF THE INVENTION

Porous biomaterials are used in a variety of implantable medicaldevices, including sutures and vascular grafts. Implants frequentlyinduce a foreign body response that results in the formation of anavascular, fibrous capsule around the implant, which limits theperformance of many implants. It has been shown that the biologicalresponse to implanted biomaterials is dependent on the microarchitectureof the biomaterial (see, e.g., Brauker et al. (1995) J. Biomed. Mater.Res. 29(12):1517-24; Sharkawy et al. (1998) J. Biomed. Mater. Res.40:586-97). There is a continuing need for porous biomaterials thatpromote vascularization in and around the implant and reduce the foreignbody reaction.

SUMMARY OF THE INVENTION

In one aspect, the invention provides porous biomaterials. The porousbiomaterials of the invention comprise a biocompatible polymer scaffolddefining an array of pores, wherein substantially all the pores have asimilar diameter, wherein the mean diameter of the pores is betweenabout 20 and about 90 micrometers, wherein substantially all the poresare each connected to at least 4 other pores, and wherein the diameterof substantially all the connections between the pores is between about15% and about 40% of the mean diameter of the pores. In someembodiments, the mean diameter of the pores is between about 30 and 40micrometers.

The biocompatible polymer scaffold of the biomaterials of the inventionmay comprise any biocompatible polymer, such as synthetic polymers,naturally-occurring polymers, or mixtures thereof. For example, thebiocompatible scaffold may be a hydrogel. In some embodiments, thebiocompatible polymer scaffold is degradable. Exemplary biocompatiblepolymers in the biocompatible polymer scaffolds include, but are notlimited to, 2-hydroxyethyl methacrylate, silicone rubber,poly(ε-caprolactone) dimethylacrylate, and collagen. Generally, thepolymer scaffold has a low level of microporosity. In some embodiments,the biomaterial of the invention has a thickness of at least 70micrometers.

The pores in the biomaterials of the invention may have any suitableshape, such as roughly or perfectly spherical. In some embodiments,substantially all the pores are connected to between about 4 to about 12other pores, such as between about 4 to about 7 other pores.

In some embodiments, the invention provides implantable devicescomprising a layer of a biomaterial. The implantable devices may be anyimplantable medical devices. The devices of the invention comprise adevice body, wherein the porous biomaterial is attached to the devicebody.

Another aspect of the invention provides methods for forming porousbiomaterials. The methods comprise the steps of: (a) forming abiocompatible polymer scaffold around a template comprising an array ofmonodisperse porogens, wherein substantially all the porogens have asimilar diameter, wherein the mean diameter of the porogens is betweenabout 20 and about 90 micrometers, wherein substantially all porogensare each connected to at least 4 other porogens, and wherein thediameter of substantially all the connections between the porogens isbetween about 15% and about 40% of the mean diameter of the porogens;and (b) removing the template to produce a porous biomaterial. Exemplaryporogens that are suitable for use in the methods of the inventioninclude, but are not limited to, polymer particles such as PMMA beadsand polystyrene beads. In some embodiments, the mean diameter of theporogens is between about 30 and 40 micrometers.

In some embodiments, step (a) of the methods for forming porousbiomaterials comprises forming the template by packing the porogens intoa mold and fusing the porogens to form connections between the porogens.In some embodiments, the template is removed by solvent extraction afterthe biocompatible polymer scaffold has been formed.

In a further aspect, the invention provides methods for promotingangiogenesis in and around an implantable biomaterial. The methodscomprise the step of implanting a porous biomaterial, wherein thebiomaterial comprises a biocompatible polymer scaffold surrounding anarray of pores, wherein substantially all the pores have a similardiameter, wherein the mean diameter of the pores is between about 20 andabout 90 micrometers, wherein substantially all pores are each connectedto at least 4 other pores, and wherein the diameter of substantially allthe connections between the pores is between about 15% and about 40% ofthe mean diameter of the pores.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of thisinvention will become more readily appreciated as the same become betterunderstood by reference to the following detailed description, whentaken in conjunction with the accompanying drawings, wherein:

FIG. 1 shows a perspective view of a representative medical device ofthe invention with a portion of the porous biomaterial layer removed toexpose the underlying device body.

FIG. 2 shows a transverse cross-section of the medical device of FIG. 1.

FIG. 3 shows a scanning electron micrograph of an exemplary porousbiomaterial of the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Unless specifically defined herein, all terms used herein have the samemeaning as they would to one skilled in the art of the presentinvention.

One aspect of the invention provides porous biomaterials. The porousbiomaterials of the invention comprise a biocompatible polymer scaffolddefining an array of pores, wherein substantially all the pores have asimilar diameter, wherein the mean diameter of the pores is betweenabout 20 and about 90 micrometers, wherein substantially all the poresare each connected to at least 4 other pores, and wherein the diameterof substantially all the connections between the pores is between about15% and about 40% of the mean diameter of the pores. In someembodiments, the biomaterial has a thickness of at least 70 micrometers.For example, the biomaterials may have a thickness between about 100 and1000 micrometers, such as between about 100 micrometers and about 500micrometers.

The polymer scaffold of the biomaterials of the invention may compriseany biocompatible polymer, such as synthetic polymers,naturally-occurring polymers, or mixtures thereof. Exemplary syntheticbiocompatible polymers include, but are not limited to, 2-hydroxyethylmethacrylate (HEMA), silicone rubber, poly(s-caprolactone)dimethylacrylate, polysulfone, (poly)methy methacrylate (PMMA), solubleTeflon-AF, (poly) ethylenetetrapthalate (PET, Dacron), Nylon, polyvinylalcohol, polyurethane, and mixtures thereof. Exemplarynaturally-occurring biocompatible polymers include, but are not limitedto, fibrous or globular proteins, complex carbohydrates,glycosaminoglycans, or mixtures thereof. Thus, the polymer scaffold mayinclude collagens of all types, elastin, hyaluronic acid, alginic acid,desmin, versican, matricelluar proteins such as SPARC (osteonectin),osteopontin, thrombospondin 1 and 2, fibrin, fibronectin, vitronectin,albumin, etc. Natural polymers may be used as the scaffold or as anadditive to improve the biocompatibility of a synthetic polymer.

The polymer scaffold may be a hydrogel. For example, the polymerscaffold may comprise a degradable hydrogel, for example by reactinglow-molecular-weight poly(ε-caprolactone) diol with an excess ofmethacryloyl chloride to give a polyester with methacrylate endgroups,and copolymerizing this compound with 2-hydroxyethyl methacrylate (HEMA)to yield a cross-linked hydrogel with hydrolyzable linkages, asdescribed in EXAMPLE 1.

The polymer scaffold generally has a low level of microporosity. Theterm “microporosity” is a measure of the presence of small microporeswithin the polymer scaffold itself (as opposed to the pores defined bythe scaffold). In some embodiments, all or substantially all of themicropores in the polymer scaffold are between about 0.1 micrometers andabout 5 micrometers, such as between about 0.1 micrometers and about 3micrometers or between about 0.1 micrometers and about 2 micrometers.The term “low level of microporosity” means that micropores representless than 2% of the volume of the polymer scaffold, as measured bymeasuring the percentage void space in a cross-section through thepolymer scaffold.

According to the invention, substantially all the pores in thebiomaterial have a similar diameter. As used herein, the term“substantially all the pores” refers to at least 90% of the pores in thebiomaterial, such as at least 95% or at least 97% of the pores. The term“diameter of a pore” refers to the longest line segment that can bedrawn that connects two points within the pore, regardless of whetherthe line passes outside the boundary of the pore.

Two pores have a “similar diameter” when the difference in the diametersof the two pores is less than about 20% of the larger diameter.Typically, the mean diameter of the pores is between about 20 and about90 micrometers, such as between about 25 and 75 micrometers or betweenabout 30 and 60 micrometers. In some embodiments, the mean diameter ofthe pores is between about 30 and 40 micrometers.

The pores may have any suitable shape. For example, the pores may beroughly, or perfectly, spherical, as shown in EXAMPLE 1. Other suitablepore shapes include, but are not limited to, dodecahedrons (such aspentagonal dodecahedons), and ellipsoids. In some embodiments,substantially all the pores have a roundness of at least about 0.1, suchas at least about 0.3 or at least about 0.7. As used herein, “roundness”is defined as (6×V)/(pi×D³), where V is the volume and D is thediameter. For example, a sphere has a roundness equal to 1.

In the array of pores, substantially all the pores are each connected toat least four other pores. In some embodiments, substantially all thepores are connected to between about 4 to 12 other pores, such asbetween about 4 to 7 other pores. Substantially all the connectionsbetween the pores have a diameter that is between about 15% and about40%, such as between about 25% and about 30%, of the mean diameter ofthe pores. As used herein, the term “diameter of the connection betweenthe pores” refers to the diameter of the cross-section of the connectionbetween two pores in the plane normal to the line connecting thecentroids of the two pores, where the plane is chosen so that the areaof the cross-section of the connection is at its minimum value. The term“diameter of a cross-section of a connection” refers to the averagelength of a straight line segment that passes through the center, orcentroid (in the case of a connection having a cross-section that lacksa center), of the cross-section of a connection and terminates at theperiphery of the cross-section. The term “substantially all theconnections” refers to at least 90% of the connections in thebiomaterial, such as at least 95% or at least 97% of the connections.

The diameter and shape of the pores, as well as the connections betweenthem may be assessed using scanning electron microscopy, as described inEXAMPLE 1. FIG. 3 shows a scanning electron micrograph of an exemplaryporous biomaterial of the invention.

The porous biomaterials of the invention allow a high degree of bloodvessel formation inside the biomaterial when implanted into a host, asdescribed in EXAMPLE 2. The biomaterials of the invention are useful formany applications, including, but not limited to, tissue engineering andrepair. For example, the biomaterials may be applied as a wounddressing, as an artificial skin substitute for the treatment of burnsand wounds. In some embodiments, hydroxyapatite is added to the polymerscaffold for use in bone repair and bone tissue engineering.

In some embodiments, the invention provides implantable devicescomprising a layer of a biomaterial, wherein the biomaterial comprises abiocompatible polymer scaffold surrounding an array of pores, whereinsubstantially all the pores have a similar diameter, wherein the meandiameter of the pores is between about 20 and about 90 micrometers,wherein substantially all pores are each connected to at least 4 otherpores, and wherein the diameter of substantially all the connectionsbetween the pores is between about 15% and about 40% of the meandiameter of the pores.

The biomaterial in or on the implantable devices of the invention are asdescribed above. In some embodiments, the layer of biomaterial has athickness of at least 70 micrometers. For example, the layer ofbiomaterial may have a thickness between about 100 and 1000 micrometers,such as between about 100 micrometers and about 500 micrometers.

The implantable devices may be any implantable medical devices,including, but not limited to, chemical sensors or biosensors (such asdevices for the detection of analyte concentrations in a biologicalsample), cell transplantation devices, drug delivery devices such ascontrolled drug-release systems, electrical signal delivering ormeasuring devices, prosthetic devices, and artificial organs. The layerof biomaterial improves the biocompatibility of the implanted medicaldevice (such as the biocompatibility and communication ofneuroelectrodes and pacemaker leads with surrounding tissues), improvesthe sealing of skin to percutaneous devices (such as in-dwellingcatheters or trans-cutaneous glucose sensors), enhances tissueintegration, and provides barriers for immunoisolation of cells inartificial organs systems (such as pancreatic cells devices), and toimprove the healing of vessels after balloon angioplasty and stentplacement.

In some embodiments, the devices of the invention comprise a devicebody, wherein the porous biomaterial is attached to the device body.Some embodiments provide medical devices comprising a device body and aporous biomaterial of the invention attached to the device body. Theporous biomaterial may be immobilized onto (or within) a surface of animplantable or attachable medical device body. In some embodiments, theporous biomaterial is attached to the outer surface of the device body.For example, the porous biomaterial may be disposed over substantiallythe entire outer surface of the device body. The modified surface willtypically be in contact with living tissue after implantation into ananimal body. As used herein, “implantable or attachable medical device”refers to any device that is implanted into, or attached to, tissue ofan animal body, during the normal operation of the device (e.g.,implantable drug delivery devices). Such implantable or attachablemedical device bodies can be made from, for example, nitrocellulose,diazocellulose, glass, polystyrene, polyvinylchloride, polypropylene,polyethylene, dextran, Sepharose, agar, starch, and nylon. Linkage ofthe porous biomaterial to a device body can be accomplished by anytechnique that does not destroy the desired properties of the porousbiomaterial. For example, a surface of an implantable or attachablemedical device body can be modified to include functional groups (e.g.,carboxyl, amide, amino, ether, hydroxyl, cyano, nitrido, sulfanamido,acetylinic, epoxide, silanic, anhydric, succinimic, azido) forimmobilizing a porous biomaterial thereto. Coupling chemistries include,but are not limited to, the formation of esters, ethers, amides, azidoand sulfanamido derivatives, cyanate and other linkages to functionalgroups available on the porous biomaterial.

In some embodiments, a surface of a device body that does not possessuseful reactive groups can be treated with radio-frequency dischargeplasma (RFGD) etching to generate reactive groups (e.g., treatment withoxygen plasma to introduce oxygen-containing groups; treatment withpropyl amino plasma to introduce amine groups). When RFGD glow dischargeplasma is created using an organic vapor, deposition of a polymericoverlayer occurs on the exposed surface. RFGD plasma deposited filmsoffer several unique advantages. They are smooth, conformal, anduniform. Film thickness is easily controlled and ultrathin films(10-1000 Angstroms) are readily achieved, allowing for surfacemodification of a material without alteration to its bulk properties.Moreover, plasma films are highly-crosslinked and pin-hole free, andtherefore chemically stable and mechanically durable. RFGD plasmadeposition of organic thin films has been used in microelectronicfabrication, adhesion promotion, corrosion protection, permeationcontrol, as well as biomaterials (see, e.g., U.S. Pat. No. 6,131,580).

Some medical devices of the invention are adapted to be implanted intothe soft tissue of an animal, such as a mammal, including a human,during the normal operation of the medical device. Implantable medicaldevices of the invention may be completely implanted into the softtissue of an animal body (i.e., the entire device is implanted withinthe body), or the device may be partially implanted into an animal body(i.e., only part of the device is implanted within an animal body, theremainder of the device being located outside of the animal body).Representative examples of completely implantable medical devicesinclude, but are not limited to: cardiovascular devices (such asvascular grafts and stents), artificial blood vessels, artificial bonejoints, such as hip joints, and scaffolds that support tissue growth (insuch anatomical structures as nerves, pancreas, eye and muscle).Representative examples of partially implantable medical devicesinclude: biosensors (such as those used to monitor the level of drugswithin a living body, or the level of blood glucose in a diabeticpatient) and percutaneous devices (such as catheters) that penetrate theskin and link a living body to a medical device, such as a kidneydialysis machine.

Some medical devices of the invention are adapted to be affixed to softtissue of an animal, such as a mammal, including a human, during thenormal operation of the medical device. These medical devices aretypically affixed to the skin of an animal body. Examples of medicaldevices that are adapted to be affixed to soft tissue of an animalinclude skin substitutes, and wound or burn treatment devices (such assurgical bandages and transdermal patches).

FIG. 1 shows a representative medical device 10 of the presentinvention, in the form of an implantable drug delivery device, whichincludes a device body 12 to which is attached a porous biomateriallayer 14. In the embodiment shown in FIG. 1, porous biomaterial layer 14has been partially removed to show device body 12 beneath. Device body12 is indicated by hatching. As shown in the cross-sectional view ofmedical device 10 in FIG. 2, porous biomaterial layer 14 includes aninternal surface 18, attached to device body 12, and an external surface20.

Due to the biocompatibility of the porous biomaterials of the inventionused in the construction of medical device 10, the presence of theporous biomaterial on the device body of a medical device will reduce oreliminate the foreign body response to the device body afterimplantation into, or attachment to, tissue of an animal body.

In some embodiments, the medical devices of the invention furthercomprise biologically active molecules within the porous biomaterialattached to the device body to provide for the controlled delivery ofdrugs and other biologically active molecules, such as DNA, RNA, orproteins. The biologically active molecules may be attached, covalentlyor non-covalently, to the crosslinking molecules or polymer molecules inthe porous biomaterial.

Any reactive functional group present on polymer molecules within theporous biomaterial can be used to covalently attach biologically activemolecules to the porous biomaterial. The following publications,incorporated herein by reference, describe examples of technologies thatare useful for attaching biologically active molecules to polymermolecules, such as the polymers present in the porous biomaterial of thepresent invention: Nuttelman et al. (2001) J. Biomed. Mater. Res.57:217-223; Rowley et al. (1999) Biomaterials 20:45-53; Hubbel (1995)Biotechnology 13:565-76; Massia & Hubbell (1990) Anal. Biochem187:292-301; Drumheller et al. (1994) Anal. Biochem. 222:380-8;Kobayashi & Ikada (1991) Curr. Eye Res. 10:899-908; Lin et al. (1992) J.Biomaterial Sci. Polym. Ed 3:217-227; and Bellamkonda et al. (1995) J.Biomed Mater. Res. 29:663-71.

The biologically active molecules may also be introduced into the porousbiomaterial by forming the porous biomaterial in the presence of thebiologically active molecules, by allowing the biologically activemolecules to diffuse into a porous biomaterial, or by otherwiseintroducing the biologically active molecules into the porousbiomaterial.

The biologically active molecules can be attached to every part of thedevice, or to only a portion of the device. For example, in someembodiments, that are adapted to be implanted into an animal,biologically active molecules that act to decrease the foreign bodyreaction (e.g., anti-inflammatory agents, and immunomodulatory agents)are attached only to the surface(s) of the device that is/are in contactwith living tissue in the animal body. The biologically active moleculesserve to decrease the foreign body reaction of the living body againstthe implanted structure.

Another aspect of the invention provides methods for forming porousbiomaterials. The methods comprise the steps of: (a) forming abiocompatible polymer scaffold around a template comprising an array ofmonodisperse porogens, wherein substantially all the porogens have asimilar diameter, wherein the mean diameter of the porogens is betweenabout 20 and about 90 micrometers, wherein substantially all porogensare each connected to at least 4 other porogens, and wherein thediameter of substantially all the connections between the porogens isbetween about 15% and about 40% of the mean diameter of the porogens;and (b) removing the template to produce a porous biomaterial.

The template used in the methods of the invention comprises an array ofporogens. As used herein, the term “porogens” refers to any structuresthat can be used to create a template that is removable after thebiocompatible polymer scaffold is formed under conditions that do notdestroy the polymer scaffold. Exemplary porogens that are suitable foruse in the methods of the invention include, but are not limited to,polymer particles such as PMMA beads and polystyrene beads.

The porogens may have any suitable shape that will permit the formationof a porous biomaterial with an array of pores, wherein substantiallyall the pores have a similar diameter, wherein the mean pore diameter isbetween about 20 and about 90 micrometers, wherein substantially allpores are each connected to at least 4 other pores, and wherein thediameter of substantially all the connections between the pores isbetween about 15% and about 40% of the mean diameter of the pores. Forexample, the porogens may be spherical, as shown in EXAMPLE 1. Othersuitable porogen shapes include, but are not limited to, dodecahedrons(such as pentagonal dodecahedons), and ellipsoids. In some embodiments,substantially all the porogens have a roundness of at least about 0.1,such as at least about 0.3 or at least about 0.7. “Roundness” is definedas described above.

Substantially all the porogens in the array have a similar diameter. Asused herein, the term “substantially all the porogens” refers to atleast 90% of the porogens, such as at least 95% or at least 97% of theporogens. As used herein, “diameter of the porogen” is defined as thelongest line segment that can be drawn that connects two points withinthe porogen, regardless of whether the line passes outside the boundaryof the porogen.

Two porogens have a “similar diameter” when the difference in thediameters of the two porogens is less than about 20% of the largerdiameter. Porogens having similar diameters of a desired size may beobtained by size fractionation, as described, for example, in EXAMPLE 1.Typically, the mean diameter of the porogens is between about 20 andabout 90 micrometers, such as between about 25 and 75 micrometers orbetween about 30 and 60 micrometers. In some embodiments, the meandiameter of the porogens is between about 30 and 40 micrometers.

In the array of porogens, substantially all the porogens are eachconnected to at least 4 other porogens. In some embodiments,substantially all the porogens are connected to between about 4 to 12other porogens, such as between about 4 to 7 other porogens.Substantially all the connections between porogens have a diameter thatis between about 15% and about 40%, such as between about 25% and about30%, of the mean diameter of the porogens. The term “substantially allthe connections” refers to at least 90% of the connections in the arrayof porogens, such as at least 95% of the connections or at least 97% ofthe connections.

In some embodiments, the invention provides methods for forming thetemplate. The methods may comprise packing the porogens into a mold. Anymold may be used for packing the porogens. For example, a suitable moldmay be formed using two glass microscope slides separated by 1-mm or3-mm spacers. The porogens may be packed into a mold using ultrasonicagitation, or any other suitable method for obtaining a closely packedarray of porogens.

In some embodiments, the methods for forming the template comprisefusing the porogens to form the connections between the porogens. Theporogens may be fused by sintering. Typically, the sintering temperatureis higher than the glass transition temperature of the polymer, such asbetween about 10° C. and about 50° C. higher than the glass transitiontemperature of the polymer. Increasing the duration of the sinteringstep at a given temperature increases the connection size; increasingthe sintering temperature increases the growth rate of the connections.Suitable sintering times are generally between 1 and 48 hours. In someembodiments, PMMA beads are sintered for 19 hours at 140° C. to produceconnection diameters of about 30% of the diameter of the beads, asdescribed in EXAMPLE 1. The porogens can also be fused by other methods.For example, the porogens can be fused by partially dissolving them bytreatment with a suitable solvent.

Once a template has been created, a biocompatible polymer scaffold isformed around the template. Suitable biocompatible polymers are asdescribed above. In some embodiments, the polymer scaffold is formedaround the template by polymerizing a polymer precursor mixture aroundthe template. The polymer precursor mixture typically comprises polymerprecursors and suitable cross-linking reagents, as described in EXAMPLE1.

After a biocompatible polymer scaffold has been formed, the template isremoved to produce the porous biomaterial. In some embodiments, thetemplate is removed by solvent extraction. For example, PMMA beads maybe extracted with a 9:1 v/v acetone-water solution, as described inEXAMPLE 1. The porous biomaterials formed using the methods of theinvention comprise an array of uniformly shaped pores, as shown in FIG.3.

In a further aspect, the invention provides methods for promotingangiogenesis in and around an implantable biomaterial. The methodscomprise the step of implanting a porous biomaterial, wherein thebiomaterial comprises a biocompatible polymer scaffold surrounding anarray of pores, wherein substantially all the pores have a similardiameter, wherein the mean diameter of the pores is between about 20 andabout 90 micrometers, wherein substantially all pores are each connectedto at least 4 other pores, and wherein the diameter of substantially allthe connections between the pores is between about 15% and about 40% ofthe mean diameter of the pores. The biomaterials used in the methods forpromoting angiogenesis are as described above. As shown in EXAMPLE 2,the porous biomaterials of the invention promote the growth of bloodvessels into the biomaterials.

The following examples illustrate representative embodiments nowcontemplated for practicing the invention, but should not be construedto limit the invention.

Example 1

This Example describes an exemplary method of the invention for makingbiomaterials with tightly controlled pore diameters.

Materials: Poly(methyl methacrylate) (PMMA) beads with diameter lessthan 100 micrometers were purchased from Kupa, Inc (Product No.BN-PW-CC). Larger beads were obtained from Polysciences (25,000 MW),along with 2-hydroxyethyl methacrylate (ophthalmic grade), ethyleneglycol, and tetraethylene glycol dimethacrylate.2,2-dimethoxy-2-phenylacetophenone was purchased from Ciba-Geigy.Poly(ε-caprolactone) diol (M_(n)˜530) and all other chemicals wereacquired from Aldrich. Non-porous silicone rubber was purchased fromInvotec Intl. (Cat. No. 20-10680). Sieves of the desired opening sizeswere custom made with stainless steel meshes purchased fromMcMaster-Carr. All materials were used as received unless otherwisespecified.

Implant Fabrication: PMMA beads were thoroughly fractionated to thedesired sizes (20±5 μm, 35±5 μm, 50±5 μm, 70±5 μm, 90±5 μm and 160±15μm) with an ATM Model L3P Sonic Sifter. Each bead size was separatedwith an iterative process, with an iteration comprising the followingsteps: (1) the beads were sieved for approximately 10 minutes; (2) thebeads were decanted from the sieves; and the fractions were labeled; (3)the monodispersity of the beads was examined by light microscopy afterattaching a monolayer of beads to a slide with transparent tape; (4) themeshes were unclogged by blowing from underneath with compressed air;and (5) the beads were replaced on the same sieves from which they wereremoved. Five to six iterations were required for each bead size toyield size fractions with the above tolerances. The beads were added tomolds consisting of two microscope slides separated by nylon spacers,packed by ultrasonic agitation, and sintered for 19 hours at 140° C. togive neck sizes of 30% of the bead diameter. These fused-bead poretemplates were infiltrated with a polymer precursor comprising thecomponents in Table 1.

TABLE 1 Polymer Precursor Mixture Component Volume (mL) 2-hydroxyethylmethacrylate (HEMA) 7.0 Ethylene glycol 2.1 Water 0.9 Tetraethyleneglycol dimethacrylate 0.31 Type I collagen in 0.02N acetic acid 0.5 (4mg/mL) Ammonium persulfate (0.4 g/mL) 0.7 Sodium metabisulfite (0.15g/mL) 0.7

After allowing the mixture to polymerize for 24 hours, the PMMAtemplates were extracted with a 9:1 v/v acetone-water solution to yieldcross-linked porous hydrogel slabs of polyHEMA doped with Type Icollagen. The slabs were punched into 4-mm disks, sterilized in 70%ethanol, rinsed every 24 hours for 1 week with endotoxin-filteredsterile water, and stored in sterile phosphate-buffered saline untiluse.

Degradable Cross-Linker Synthesis: Methacryloyl chloride was distilledat 30 mm Hg and 24° C. Then 4.0 g of vacuum-dried poly(ε-caprolactone)diol was dissolved in 34 mL of dry methylene chloride. Residual moisturewas removed with molecular sieves. The solution was added to a 250-mLflask containing a dry nitrogen atmosphere at 0° C. Next, 1.25 molarexcess (2.78 mL) of triethylamine was added to the flask, followed bydropwise addition of 1.25 molar excess (1.92 mL) of freshly distilledmethacryloyl chloride diluted with 6 mL of dry methylene chloride. Thereaction mixture was stirred for 8 hours at 0° C. The precipitated salt(triethylamine hydrochloride) was filtered off. Then unreactedtriethylamine was removed by extracting with 1% hydrochloric acid untilthe aqueous layer remained colorless. Next, any methacrylic acid wasremoved by extracting with 3% sodium hydroxide until the aqueous layerremained colorless. The excess base was removed by repeated rinses withdeionized water. The methylene chloride was evaporated to yield a whitepaste that melts into a colorless, viscous liquid at 25° C. The puritywas verified by ¹H-NMR and infrared spectroscopy.

Degradable Porous Hydrogel Preparation: HEMA monomer was purified bydiluting 1:1 with water and extracting 4 times with hexane. Sodiumchloride was then added until saturation, so that the HEMA separated outas the top layer. The HEMA layer was dried with sodium sulfate afterdiluting 1:1 with methylene chloride. Finally, the methylene chloridewas evaporated. The monomer was verified to be dimethacrylatefree bypolymerizing it into a linear hydrogel and solubilizing in 1:1 v/vacetone-methanol.

PMMA fused-bead pore templates (described earlier) were infiltrated witha polymer precursor consisting of the components in Table 2, and themixture was irradiated with 365 nm UV light (UVP, Model UVGL-25) for 45minutes. Then the pore templates were removed as described earlier, andthe porous gels were verified to degrade completely within 2 weeks in 1M sodium hydroxide at 37° C.

TABLE 2 Degradable Hydrogel Precursor Mixture Component Volume (mL)Purified 2-hydroxyethyl methacrylate (HEMA) 7.0 mL Ethylene glycol 2.1mL Water 0.9 mL Ethanol 0.02 mL Poly(ε-caprolactone) dimethacrylate 0.62mL Type I collagen in 0.02N acetic acid 0.5 mL (4 mg/mL)2,2-dimethoxy-2-phenylacetophenone 0.07 g

Scanning Electron Microscopy: Scanning Electron Microscopy (SEM) imageswere acquired with an FEI Sirion field emission electron microscope withno sample coatings and an accelerating voltage of 1 kV.

Results: All peaks in the 200-Hz proton NMR spectrum of the synthesizedpoly(ε-caprolactone dimethacrylate) are consistent with a successfulhigh-yield synthesis. For many of the hydrogen types, there are twopeaks, with the secondary peak shifted slightly downfield from theprimary peak; this is especially noticeable for hydrogen types a and b.The primary peak corresponds to the case where there are two or morecaprolactone units on the end of the molecule, while the smaller peakcorresponds to the less abundant structure with one caprolactone unit onthe end.

Prior to assembly of the templates, the monodispersity of each bead sizeof the PMMA beads was examined by light microscopy. For each bead size,at least 95% of the beads had a diameter equal to the mean bead diameter+/−20%.

The monodispersity of the necks in the porogen templates was measuredfrom SEM images. For each bead size, at least 95% of the necks wereverified to be between 25% and 35% of the mean bead diameter. ScanningElectron Microscopy was also used to determine the structure of theporous biomaterial. Examination of the SEM images revealed that eachpore was connected to between 4 and 7 neighboring pores. The porousmaterials had a precisely uniform pore structure, as shown in FIG. 3.

Example 2

This Example describes an in vivo analysis of the correlation betweenpore size in the biomaterials of the invention and the promotion ofvascular in-growth.

Materials: Collagen and all antibodies were bought from BD Biosciences,mounting media (Permount) from Fisher.

Implantation and Histology: In experiment 1, disks (4-mm diameter, 1-mmthickness) of each of three porous materials (35-, 70-, and 160-μm porediameter) made according to the method described in Example 1, alongwith a non-porous silicone rubber (Invotec Intl., Cat. No. 20-10680),were implanted subcutaneously into the dorsum of 7-month-old female mice(n=9). After 4 weeks, the implants were explanted, stabilized withmethyl Carnoy's fixative, embedded in paraffin, cross-sectioned at thewidest point, stained with an anti-mouse panendothelial cell antibody(MECA-32) in combination with biotinylated goat anti-rat IgG secondaryantibody and avidin-biotin complex, developed with diaminobenzidine, andcoverslipped with Permount. Sections were viewed in brightfield with a4× objective, and imaged digitally. All intraimplant blood vesselprofiles in each section were counted manually.

After analyzing results from experiment 1, materials with nominal porediameters of 20, 35, 50, 70, and 90 μm were implanted in 3-month-oldmale mice and analyzed similarly (35-, 50-, 70-, and 90-μm pore sizes inexperiment 2, and 20- and 35-μm pore sizes in experiment 3, with n=7animals per group).

Results: As Table 3 illustrates, the number of blood vessels inside thepores increases as the pore diameter is decreased, reaching a maximum ata pore size of ˜35 μm. The 160-μm pore diameter material was almostcompletely avascular. The smoothly decreasing trend from 35 to 160 μmsuggests a dose response, with the number of vessels roughlyproportional to the internal surface area (which is inverselyproportional to the pore diameter). Results from a separate in vivoexperiment, where the collagen-doped 35-μm pore material was compared toa protein-free control, indicate that the materials are vascularized tothe same extent regardless of whether protein is added. Ostensibly, theobserved dose response is caused by the presence of the surface per serather than by the protein additives in the surface.

The 20-μm pores showed a significant decrease in vessel density relativeto the 35-μm pores. This may be due to the inability of host macrophagecells or fibroblasts to penetrate deeply into the smaller pore material,as these two materials have uniformly-sized pore throat diameters of 6and 10 μm respectively. Macrophages, which are about 10 μm in size, areexpected to be slowed substantially by 6-μm constrictions. This issupported by examination of the number of vessels as a function of depthwithin the implant. Materials with pore sizes 50 μm or larger hadapproximately the same vessel density in the core of the implant as theydid near the perimeter, while the 35-μm pore material had much lowervessel density in the core; the 20-μm material generally had highvascular density near the surface, but was completely vessel-free in allcases at the implant core.

TABLE 3 Effect of Pore Diameter on Blood Vessel Density Spherical PoreBlood Vessel Density Diameter Experimental (Mean Vessel Profiles/(micrometers) Group mm² ± standard error) 20 3  72 ± 24 35 1 257 ± 40 2226 ± 34 3 157 ± 18 50 2 180 ± 15 70 1 125 ± 23 2 126 ± 25 90 2  75 ± 10160 1  9 ± 1

A depth-profiled intra-implant vessel density as a function of porediameter is shown in Table 4 for experiment 1, and in Table 5 forcombined experiments 1, 2, and 3. The 35-μm pore material inducedmaximum number of intra-plant blood vessels. For 50-μm pores and larger,the vessel density is almost uniformly distributed through the entire1-mm thickness of the implant.

TABLE 4 Effect of Pore Diameter on Blood Vessel Density at DifferentDepths for Experiment 1 Spherical Pore Depth from Blood Vessel DensityDiameter Implant Surface (Mean Vessel Profiles/ (micrometers)(micrometers) mm² ± standard error) 35  0-100  323 ± 100 100-200 308 ±97 200-300 276 ± 91 300-400 232 ± 89 400-500 168 ± 63 70  0-100 109 ± 60100-200 125 ± 49 200-300 137 ± 45 300-400 136 ± 47 400-500 114 ± 38 160 0-100 10 ± 3 100-200  9 ± 5 200-300  9 ± 3 300-400  8 ± 3 400-500  7 ±2

TABLE 5 Effect of Pore Diameter on Blood Vessel Density at DifferentDepths for Combined Experiments 1, 2, and 3 Spherical Pore Depth fromBlood Vessel Density Diameter Implant Surface (Mean Vessel Profiles/(micrometers) (micrometers) mm² ± standard error) 20  0-100  86 ± 27100-200 104 ± 34 200-300  96 ± 37 300-400  66 ± 29 400-500  41 ± 27 35 0-100 246 ± 23 100-200 245 ± 23 200-300 217 ± 24 300-400 203 ± 22400-500 178 ± 21 50  0-100 176 ± 23 100-200 188 ± 17 200-300 175 ± 19300-400 173 ± 18 400-500 172 ± 25 70  0-100 110 ± 17 100-200 130 ± 17200-300 137 ± 17 300-400 133 ± 17 400-500 120 ± 15 90  0-100 86 ± 7100-200  87 ± 10 200-300  73 ± 14 300-400  77 ± 10 400-500  64 ± 11 160 0-100 10 ± 3 100-200  9 ± 5 200-300  9 ± 3 300-400  8 ± 3 400-500  7 ±2

The mean vessel density as a function of pore diameter is shown in Table6. These data can be fit (R²=0.998) with an expression of the form:

N _(vessels) =k ₁ /d _(pore) minus k₂ /d ² _(throat) minus k₀

where N_(vessels) is the vessel density, k₁, k₂, and k₀ are fittingconstants, d_(pore) is the spherical pore diameter, and d_(throat) isthe pore throat diameter.

TABLE 6 Mean Vessel Density as a Function of Pore Diameter SphericalPore Blood Vessel Density Diameter (Mean Vessel Profiles/ (micrometers)mm² ± standard error) 20  70 ± 26 35 217 ± 22 50 176 ± 18 70 126 ± 15 90 75 ± 10 160  9 ± 2

The mean vessel density as a function of surface area is shown in Table7. The specific surface area (i.e., the surface area per unit volume) isinversely proportional to pore size and can be easily calculated whenthe average pore size and pore geometry are known. When the data areplotted as a function of specific surface area, the curve fit takes theform of a second order parabola.

TABLE 7 Mean Vessel Density as a Function of Surface Area SpecificSurface Blood Vessel Density Area (Mean Vessel Profiles/ (cm²/cm³) mm² ±standard error) 217  9 ± 2 387  75 ± 10 497 126 ± 15 696 176 ± 18 994217 ± 22 1740  70 ± 26

While the preferred embodiment of the invention has been illustrated anddescribed, it will be appreciated that various changes can be madetherein without departing from the spirit and scope of the invention.

1. A biomaterial, comprising a crosslinked biocompatible polymerscaffold defining an array of pores, wherein substantially all the poreshave a similar diameter, wherein the mean diameter of the pores isbetween about 20 and about 90 micrometers, wherein substantially all thepores are each connected to at least 4 other pores, and wherein thediameter of substantially all the connections between the pores isbetween about 15% and about 40% of the mean diameter of the pores. 2.The biomaterial of claim 1, wherein the mean pore diameter is betweenabout 30 and about 40 micrometers.
 3. The biomaterial of claim 1,wherein the biocompatible polymer scaffold is biodegradable.
 4. Thebiomaterial of claim 1, wherein the biocompatible polymer scaffold is ahydrogel.
 5. The biomaterial of claim 1, wherein the biocompatiblepolymer scaffold comprises 2-hydroxyethyl methacrylate.
 6. Thebiomaterial of claim 1, wherein the biocompatible polymer scaffoldcomprises poly(ε-caprolactone) dimethylacrylate.
 7. The biomaterial ofclaim 1, wherein the biocompatible polymer scaffold comprises collagen.8. The biomaterial of claim 1, wherein the biocompatible polymerscaffold comprises silicone rubber.
 9. The biomaterial of claim 1,wherein the biomaterial has a thickness of at least 70 micrometers. 10.An implantable device, comprising a layer of a biomaterial, wherein thebiomaterial comprises a crosslinked biocompatible polymer scaffoldsurrounding an array of monodispersed pores, wherein substantially allthe pores have a similar diameter, wherein the mean diameter of thepores is between about 20 and about 90 micrometers, whereinsubstantially all pores are each connected to at least 4 other pores,and wherein the diameter of substantially all the connections betweenthe pores is between about 15% and about 40% of the mean diameter of thepores.
 11. The implantable device of claim 10, wherein the layer ofbiomaterial has a thickness of at least 70 micrometers.
 12. The deviceof claim 10, wherein the device comprises a device body, wherein thelayer of biomaterial is attached to the device body.
 13. The device ofclaim 12, wherein the layer of biomaterial is attached to the outersurface of the device body.
 14. The device of claim 12, wherein thedevice is a medical device.
 15. A method for forming a biomaterial,comprising the steps of: (a) forming a crosslinked biocompatible polymerscaffold around a template comprising an array of monodisperse porogens,wherein substantially all the porogens have a similar diameter, whereinthe mean diameter of the porogens is between about 20 and about 90micrometers, wherein substantially all porogens are each connected to atleast 4 other porogens, and wherein the diameter of substantially allthe connections between the porogens is between about 15% and about 40%of the mean diameter of the porogens; and (b) removing the template toproduce a porous biomaterial.
 16. The method of claim 15, wherein theporogens are spherical beads.
 17. The method of claim 15, wherein theporogens comprise poly(methyl) methacrylate.
 18. The method of claim 15,wherein the biocompatible polymer scaffold comprises 2-hydroxyethylmethacrylate.
 19. The method of claim 15, wherein the biocompatiblepolymer scaffold comprises poly(ε-caprolactone) dimethylacrylate. 20.The method of claim 15, wherein the biocompatible polymer scaffoldcomprises collagen.
 21. The method of claim 15, wherein thebiocompatible polymer scaffold comprises silicone rubber.
 22. The methodof claim 15, wherein the biomaterial has a thickness of at least 70micrometers.
 23. The method of claim 15, wherein step (a) comprisesforming the template by packing the porogens into a mold and fusing theporogens to form the connections between the porogens.
 24. The method ofclaim 23, wherein the porogens are fused by sintering.
 25. A method forpromoting angiogenesis in and around an implantable biomaterial,comprising the step of implanting a porous biomaterial, wherein thebiomaterial comprises a crosslinked biocompatible polymer scaffoldsurrounding an array of pores, wherein substantially all the pores havea similar diameter, wherein the mean diameter of the pores is betweenabout 20 and about 90 micrometers, wherein substantially all pores areeach connected to at least 4 other pores, and wherein the diameter ofsubstantially all the connections between the pores is between about 15%and about 40% of the mean diameter of the pores.